Genetically engineered bacterium of Escherichia coli and method for fermentation production of L-theanine thereof

Abstract

The present invention belongs to the bioengineering field, and relates to a method for fermentation production of L-theanine by using an Escherichia coli genetically engineered bacterium. The engineered bacterium is obtained by serving a strain as an original strain, wherein the strain is obtained after performing a single copy of T7RNAP, a dual copy of gmas, xylR knockout, and sucCD knockout on an Escherichia coli W3110 genome, and by integrating genes xfp, pta, acs, gltA, and ppc, and knocking out ackA on the genome. The present invention has a high yield, and stable production performance; after 20-25 h, L-theanine has a titer of 75-80 g/L, and the yield is up to 52-55%. The fermentation broth is purified by membrane separation in combination with a cation-anion resin series technique. Moreover, the one-step crystallization yield is 72.3% and the L-theanine final product has a purity of 99%.

What is claimed is: 
     
       1. A genetically engineered bacterium producing L-theanine, wherein the genetically engineered bacterium is obtained by using a strain as an original strain, wherein the original strain is obtained after integrating a single copy of a RNA polymerase gene T7RNAP, a dual copy of a γ-glutamylmethylamide synthetase gene gmas, and a knockout of a xylose operon transcription factor gene xylR and a knockout of a succinyl-CoA synthetase gene sucCD on a genome of  Escherichia coli  W3110, and then by integrating an exogenous fructose 6-phosphate phosphoketolase gene xfp, an exogenous phosphoacetyl transferase gene pta, an exogenous acetyl-CoA synthetase gene acs, an exogenous citrate synthase gene gltA, and an exogenous phosphoenolpyruvate carboxylase gene ppc on the genome, and knocking out an acetokinase gene ackA. 
     
     
       2. The genetically engineered bacterium of  claim 1 , wherein the fructose 6-phosphate phosphoketolase gene xfp comprising the nucleotide sequence as shown in SEQ ID NO: 1; the phosphoacetyl transferase gene pta comprising the nucleotide sequence as shown in SEQ ID NO:2; the acetyl-CoA synthetase gene acs comprising the nucleotide sequence as shown in SEQ ID NO:3; the citrate synthase gene gltA comprising the nucleotide sequence as shown in SEQ ID NO:4; the phosphoenolpyruvate carboxylase gene ppc comprising the nucleotide sequence as shown in SEQ ID NO:5; and the acetokinase gene ackA comprising the nucleotide sequence as shown in SEQ ID NO:6. 
     
     
       3. The genetically engineered bacterium of  claim 1 , wherein genes xfp, pta, acs, gltA, and ppc are respectively controlled by a trc promoter.